Mining Of Natural Products From Three Strains Of Fungi

Natural Products(NPs)are a class of small molecular substances with diverse structures and biological activities.Fungal NPs have been widely used in many fields,such as medical treatment,agriculture and manufacturing,and have important research value and development prospects.Over the past two decades,Genome Mining has became an effective strategy to exploit NPs in fungi and to unravel the biosynthetic mechanisms of NPs,especially for fungal silent BGCs under laboratory conditions.Genome Mining maximizes the potential of fungal secondary metabolites.NPs and their derivatives provide a rich source for the semisynthesis or synthesis pathway of new therapeutic drugs.At present,NPs and their derivatives account for nearly 70%of small molecule drugs in clinical practice.This study was based on the bioinformatics analysis of genome sequences of Schizophyllum commune H4-8,Colletotrichum higginsianum ACCC37053 and Penicillium roqueforti FM164.To explore the NPs of three fungi,we used Genome Mining strategy,including express gene clusters in heterologous hosts of Aspergillus nidulans A1145 and Aspergillus oryzae RIB40 and overexpress Transcriptional Factor(TF).Five compounds,which could not be synthesized under conventional laboratory conditions,were isolated and characterized,and their biological activities were explored.The main results are as follows:1.Compound 1 was isolated and purified by large-scale fermentation of S.schizophyllum on PDA medium at 37?.The molecular formula of compound 1 is C₂₀H₃₀O₄and molecular weight is 334,it has the maximum UV absorption at 195 nm.Through 1-D and 2-D NMR spectrum,the structure of compound 1 was characteried as schizostatin,which is an isoprene/terpene compound with cholesterol-lowering efficacy.Preliminary antimicrobial tests showed that compound 1 had obvious inhibitory effects on Mycobacterium tuberculosis and Staphylococcus aureus,and slight inhibitory effects on Pichia pastoris and Bacillus subtilis.Three Biosynthesis Gene Clusters(BGCs)of S.schizophyllum that may encode NPs of isoprene/terpene in the genome were heterologously expressed in A.nidulans.We hope to further reveal the biosynthetic mechanism of compound 1.2.A predicted Polyketide Synthase-Nonribosomal Peptide Synthetase(PKS-NRPS)gene cluster ACCC12.1 of C.higginsianum was heterologously expressed in A.nidulans.Two compounds(2 and 3)were obtained.The molecular formula of compound 2 is C₁₅H₂₇NO₃and molecular weight is 269,it has the maximum UV absorption at 216 nm;the molecular formula of compound 3 is C₂₀H₃₇NO₂and molecular weight is 323,it has the maximum UV absorption at 219 nm.1-D and 2-D NMR spectrum showed that compound 2 and 3 were two new compounds.The antibacterial experiments showed that compound 2 had a slight inhibitory effect on M.tuberculosis,S.aureus and B.subtilis,while compound 3 had a slight inhibitory effect on B.subtilis and an obvious inhibitory effect on M.tuberculosis.The results of RT-PCR suggested that both the core gene and the modifier gene were successfully transcripted.The core gene was also expressed in A.oryzae.Only compound 2 was produced.It was speculated that compound 2 and 3 were not the true products of core genes,and the real products may be modified by A.oryzae and A.nidulans,so that the downstream modifier gene cannot play a role.Compared with its similar gene cluster,three new genes were added and co-expressed in A.nidulans,to explore the unknown function of the modifier gene in the gene cluster.However,no new compound was produced.3.Two compounds(4 and 5)were produced by overexpressing TF within a putative Highly Reducing Polyketide Synthase(HR-PKS)gene cluster in P.roqueforti.The molecular weight of compound 4 was 362,and the maximum UV absorption was found at 216 nm,while the molecular weight of compound 5 was 643,and the maximum UV absorption was found at 217 nm and 332 nm.In this study,five compounds,including schizostatin,were isolated and characterized from three fungi,and the compounds showed different anti-bacterial activites,the results indicate that Genome Mining is a effective strategy in discovery of fungal NPs.